Research Communications Microbially-Enhanced Chemisorption of Heavy Metals: A Method for the Bioremediation of Solutions Containing Long-Lived Isotopes of Neptunium and Plutonium LYNNE E. MACASKIE* AND GABRIELA BASNAKOVA School of Biological Sciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Immobilized cells of a Citrobacter sp. removed neptunium and plutonium negligibly from solution using an established technique that used biologically-produced phosphate ligand (Pi) for metal phosphate bioprecipitation. Removal of these transuranic radionuclides was enhanced by prior exposure of the biomass to lanthanum in the presence of organophosphate substrate to form cell-bound LaPO4. Polyacrylamide gel-immobilized cells removed little Np and Pu per se, but preloaded LaPO4 promoted the removal of Np and Pu upon subsequent challenge in a flowthrough column. Approximately 2 µg of Np was loaded per 1 mL, column, when the experiments were stopped after 10 mL, with maintenance of approximately 90% removal of the input metal. Transuranic element removal by this technique, generically described as microbially-enhanced chemisorption of heavy metals (MECHM), is via a hybrid of bioaccumulative and chemisorptive mechanisms.
Introduction Application of biotechnology to uranium removal from wastewater is well-established (1-4), but little attention has been paid to the removal of long-lived, R-emitting transuranic elements (5). Simple chemical precipitation is the easiest and most economical method, but the low bulk metal concentrations and formation of poorly-settling colloidal precipitates may limit the effectiveness of this approach. Uptake of 241Am was shown by Escherichia coli (6, 7) and by a Citrobacter sp. (3, 8, 9), this removing many heavy metals by metal phosphate precipitation via the release of phosphate ligand (8, 9) produced by the activity of a radioresistant (3) cell-bound phosphatase (3, 8, 9). In contrast to Am, the removal of Np and Pu is problematic. Previous studies using Citrobacter (3, 8) and other organisms (10) gave little removal of 237Np. Similarly, attempts to remove 239Pu gave only 50% removal at steady state under conditions where the removal of 241Am was complete (8). The recalcitrance of Np is attributable to the solution chemistry of the pentavalent actinide species. Np(V), the most common species in neutral solution (NpO2+), does not form insoluble phosphates (although HNpO2PO4 is insoluble; 11). Plutonium, normally Pu(IV) in solution (12, 13) is thought * Corresponding author Tel: 0044-121-414-5889; Fax: 0044-121414-6557; e-mail:
[email protected]. 184
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 1, 1998
to predominate instead as Pu(V) in natural waters (14), behaving similarly to Np(V). Np(V), although almost completely disproportionated into Np(IV) and Np(VI) in 6 M HNO3, forms stable Np(V) at neutral pH, although Np(IV) is stabilized in solutions containing complexing ligands (15) that can occur (e.g., EDTA, citrate) in wastes (16). Most waste inventories pay scant attention to the metal speciation, and this will vary according to the conditions (redox potential, pH, radiolysis, UV light) and the composition of the background ionix matrix. Hence we set up a model matrix for use in a preliminary study. A previous study using a ‘surrogate’ mixture of La(III), Th(IV), and U(VI) as a model (17) for AnIII, -(IV), and -(VI) species established that Th(IV) was removed poorly (17, 18) under conditions where La(III) and U(VI) were removed efficiently using polyacrylamide gel (PAG)-immobilized cells in a flow-through reactor (17). The recalcitrance of Th was attributed to the amorphous nature of the thorium phosphate (17, 18) and to the low availability of free Th4+ for phosphate precipitation in the presence of citrate, routinely incorporated to suppress actinide hydrolysis (19) and, in the case of Pu4+, to suppress the formation of polymeric species (20, 21). Th and Pu were removed more efficiently in the presence of La3+ with the formation of a co-crystal of lanthanum/thorium phosphate (17). This is in accordance with a generic model (microbially-enhanced chemisorption of heavy metals, MECHM) that postulates that predeposition of the phosphate crystal of a ‘benign’ metal promotes the subsequent deposition of a metal that does not precipitate easily (3, 8). The study (17) was unable to distinguish between an intercalation mechanism and simple addition of Th(HPO4)2 onto an already-formed, ‘nucleating’ deposit of LaPO4; this could be difficult to confirm by solid-state methods in the absence of published data for reference compounds. In the case of Pu and Np, the use of sufficient metal to produce enough solid for analysis presents difficulties in handling and final disposal of the active nuclides. Also, crystallographic studies are problematic; growth of sufficiently large reference crystals is very difficult (11). Regardless of the actual mechanism of MECHM (intercalative or additive), the principle of this states that the removal of a recalcitrant metal is promoted in the presence of a previously deposited metal phosphate. This was tested here, using La as the ‘priming’ metal in columns containing immobilized cells of Citrobacter sp. that were subsequently challenged with the R-emitting isotopes 237Np or 239Pu ‘spiked’ with the high-active tracer isotopes 239Np (γ-emitter) and 241Pu (β-emitter), respectively. In addition to 237Np and 239Pu, the latter isotope can contribute much of the radioactive burden to fresh wastewater (22).
Materials and Methods Cell Growth and Immobilization. The Citrobacter sp. strain N14 (used under license from Isis Innovation, Oxford, U.K.) was grown and immobilized in PAG as described previously (17, 18). Each preparation (5 g fresh weight of biomass) was divided into 50 replicate columns (working volume 3 mL; Pierce) each containing 0.1 g wet weight of biomass in 1 g of shredded material, held within the column by porous frits (Pierce) below and above the gel (bed volume, approximately 1 mL). Column Priming with a Deposit of LaPO4 and Column Testing with La. The columns were challenged with priming solution (room temperature), comprising 50 mM MOPS/ S0013-936X(97)00852-3 CCC: $14.00
1997 American Chemical Society Published on Web 01/01/1998
TABLE 1. Radioisotopes Used in This Studya isotope 239Pu 241Pu
[241Pu 237Np 239Np
half-life and emission
concn in challenge (mass)
concn in challenge (radioactivity)
2.44 × 104 yr (R) 14.5 yr (β) 14.5 yr(β) 2.10 × 106 yr (R) 2.33 day (γ)
39.30 nM 0.18 nM 1.80 nM 1.0 µm ND
21.6 Bq/mL 188 Bq/mL 1686 Bq/mL] 6.3 Bq/mL 360 counts min-1 mL-1
a Initial tests in brackets used 0.1 mL of stock 239Pu and 0.2 mL of Pu in a final volume of 10 mL. Subsequent studies (for more convenient handling) used 0.1 mL of 239Pu and 0.02 mL of 241Pu; the final concentration (239Pu + 241Pu) was 39.5 nM in a final carrier of 24 mM NO3-. The volume of 237Np taken (0.05 mL) was supplemented with 0.05 mL of 239Np (concentrated stock solution was 1200 counts s-1 mL-1; the mass concentration was not determined, ND). The total Np taken was 0.1 mL in a final concentration of NO3- in the challenge solution of 80 mM. 241
NaOH buffer/2 mM citrate buffer (pH 7), 5 mM glycerol 2-phosphate, and 1 mM lanthanum nitrate. La and citrate were mixed first. The columns were challenged (50 mL upflow; Watson Marlow Flow Inducer) at a flow rate (F) that gave an appropriate steady-state removal of La (ca. 80% or 23% removal of the input La as described), with La removal monitored by assay of inflow and outflow solutions using arsenazo III (17, 25). The primed columns were stored at 4 °C, statically, in La test solution, which comprised 10 µM La(NO3)3 in a ‘carrier’ flow of 50 mM MOPS/NaOH/0.5 mM citrate buffer, pH 7, and 5 mM glycerol 2-phosphate (10 mL). Assay of residual La was done with arsenazo III as above. Preparation of Transuranic Element Solutions. 237Np (bulk, R-isotope), separated from its high-active β-daughter 233Pa, and 239Np (γ-tracer) were gifts from BNFL. All Np experiments were done within 3 days. 239Pu (bulk, R-isotope) and 241Pu (β-tracer) were from AEA Fuel Services, Harwell, U.K. The metals were supplied in carriers of 8 M (Np), 2 M (239Pu), or 1 M HNO3 (241Pu) and when prepared in test challenge solutions (below) were neutralized just prior to use with NaOH (4 M) to give a final pH of approximately 7. Column challenge solution was prepared fresh by introduction of stock actinides (in HNO3) into a polypropylene scintillation vial. To this was added, in order, 1 mL of 5 mM trisodium citrate/citric acid buffer, pH 6.9; 2.5 mL of 200 mM MOPS/NaOH buffer, pH 7; 0.1 mL of 500 mM glycerol 2-phosphate (sodium salt); and 6.4 mL of distilled water and the appropriate volume of NaOH. The final volume was made to 10 mL with water; the final actinide concentrations were approximately 40 nM (Pu) and 1 µM (Np) (Table 1). Valence testing (for Np; Pu was assumed to be similar) of the carrier solution was done by paper chromatography with radioactive spots (239Np γ-tracer) visualized using a phosphorImager (3, 23). The Rf values of Th(IV) and U(VI) for reference were determined in parallel and visualized by spraying with arsenazo III (composition as in ref 17) since the specific γ-activity of these was very low at convenient mass loadings (3). Challenge of the Immobilized Cells with Actinide Elements. The column was prepared as described previously (8, 24). For use, the columns were taken from storage, and a cotton wool filter (0.5 g of nonabsorbent cotton wool in the barrel of a Pasteur pipet) was inserted at the distal end to trap any outflow particulate species. Columns that had been primed and tested against 10 µM La were drained and challenged with a flow (downward, using a ‘pull’ pump to maintain negative pressure in the column) of metal-unsupplemented test solution (F ) 6 mL/h, 10 mL), until the top of the bed was just visible above the meniscus. Freshly prepared test actinide solution (above) was introduced, and the column outflow solution was collected in approximately
FIGURE 1. Columns of PAG-immobilized cells of Citrobacter sp. were challenged with solutions of Pu (filled symbols) or Np (open symbols) as shown in Table 1 in challenge solution as described in the text, either without prior exposure to La (), () or following priming at an efficiency (set by the flow rate of the priming solution through the column) of 77% (for subsequent challenge with Np, O) or 83% (for subsequent challenge with Pu, b) removal of the input La. A second set of experiments reduced the level of priming to 21% (for subsequent challenge with Np, 4) and 24% (for subsequent challenge with Pu, 2) removal of the input La. Determination of the transuranic element concentration in the column inflow and exit solutions was by γ-counting or by β-counting for the probes 239Np and 241Pu as described in the text. 2-mL fractions, with the volume estimated by weight. The Np and Pu contents of the input and outflow solutions were determined by γ-counting (for 239Np: Canberra high resolution germanium detector connected to a Nuclear Data multichannel analyzer) or by scintillation counting of 241Pu in 10 mL of Hisafe II scintillation cocktail (Pharmacia) in a TriCarb 2700 liquid scintillation analyzer (Packard Instrument Company, Meriden CT).
Results and Discussion Neptunium migrated at Rf positions corresponding to U(VI) and Th(IV) and between these. The valence was suggested to be a mixture of species, but the exact speciation in the tests would depend on the time taken to reach the disproportionation equilibrium of Np(V) within the run time of each column (
